The mechanism of photo-oxidation of tryptophan (Trp) sensitized by riboflavin (RF) was examined employing high concentrations of Trp and RF, with a high intensity 365 nm light emitting diode (LED) ...source under N2, 20% and 100% O2 atmospheres. Dimerization of Trp was a major pathway under the N2 atmosphere, though this occurred with a low yield (DφTrp = 5.9 × 10−3), probably as a result of extensive back electron transfer reactions between RF•- and Trp(H)•+. The presence of O2 decreased the extent of this back electron transfer reaction, and the extent of Trp dimerization. This difference is attributed to the formation of O2•- (generated via electron transfer from RF•- to O2) which reacts rapidly with Trp• leading to extensive consumption of the parent amino acid and formation of peroxides and multiple other oxygenated products (N-formylkynurenine, alcohols, diols) of Trp, as detected by LC-MS. Thus, it appears that the first step of the Type 1 mechanism of Trp photo-oxidation, induced by this high intensity 365 nm light source, is an electron transfer reaction between the amino acid and 3RF, with the presence of O2 modulating the subsequent reactions and the products formed, as a result of O2•- formation. These data have potential biological significance as LED systems and RF-based treatments have been proposed for the treatment of pathological myopia and keratitis.
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•Riboflavin (RF)-mediated photoxidation using LEDs has therapeutic potential.•Oxidation of Trp sensitized by RF-LED (365 nm) was studied under N2 and O2 atmospheres.•Back electron transfer and Trp dimerization are major reaction pathways under N2.•O2 decreases Trp dimerization, and gives oxygenated products via O2•- formation.•Trp photo-oxidation by RF-LED mainly involves a type 1 mechanism under N2 and O2.
Disulfide bonds play a key function in determining the structure of proteins, and are the most strongly conserved compositional feature across proteomes. They are particularly common in extracellular ...environments, such as the extracellular matrix and plasma, and in proteins that have structural (e.g. matrix) or binding functions (e.g. receptors). Recent data indicate that disulfides vary markedly with regard to their rate of reaction with two-electron oxidants (e.g. HOCl, ONOOH), with some species being rapidly and readily oxidized. These reactions yielding thiosulfinates that can react further with a thiol to give thiolated products (e.g. glutathionylated proteins with glutathione, GSH). Here we show that these ‘oxidant-mediated thiol-disulfide exchange reactions’ also occur during photo-oxidation reactions involving singlet oxygen (1O2). Reaction of protein disulfides with 1O2 (generated by multiple sensitizers in the presence of visible light and O2), yields reactive intermediates, probably zwitterionic peroxyl adducts or thiosulfinates. Subsequent exposure to GSH, at concentrations down to 2 μM, yields thiolated adducts which have been characterized by both immunoblotting and mass spectrometry. The yield of GSH adducts is enhanced in D2O buffers, and requires the presence of the disulfide bond. This glutathionylation can be diminished by non-enzymatic (e.g. tris-(2-carboxyethyl)phosphine) and enzymatic (glutaredoxin) reducing systems. Photo-oxidation of human plasma and subsequent incubation with GSH yields similar glutathionylated products with these formed primarily on serum albumin and immunoglobulin chains, demonstrating potential in vivo relevance. These reactions provide a novel pathway to the formation of glutathionylated proteins, which are widely recognized as key signaling molecules, via photo-oxidation reactions.
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•Disulfide bonds (DSBs) are critical to protein structure and function.•DSBs are rapidly oxidized by singlet oxygen and other oxidants to reactive species.•These DSB-derived intermediates react with GSH to give glutathionylated proteins.•Glutathionylation can be diminished by reductants, but does not repair DSB damage.•Oxidation of human plasma DSBs gives glutathionylated albumin and immunoglobulins.
•First report of circulating mtDNA-cn in people with Type 1 diabetes and non-diabetic subjects.•Lower mtDNA-cn in adults with vs. without Type 1 diabetes.•Lower mtDNA-cn in adults with Type 1 ...diabetes with vs. without chronic kidney disease.•mtDNA-cn levels associated negatively with smoking, inflammation and blood pressure.
Mitochondrial damage is implicated in diabetes pathogenesis and complications. Mitochondrial DNA copy number (mtDNA-cn) in human Type 1 diabetes (T1D) studies are lacking. We related mtDNA-cn in T1D and non-diabetic adults (CON) with diabetes complications and risk factors.
Cross-sectional study: 178 T1D, 132 non-diabetic controls. Associations of whole blood mtDNA-cn (qPCR) with complications, inflammation, and C-peptide.
mtDNA-cn (median (LQ, UQ)) was lower in: T1D vs. CON (271 (189, 348) vs. 320 (264, 410); p < 0.0001); T1D with vs. without kidney disease (238 (180, 309) vs. 294 (198, 364); p = 0.02); and insulin injection vs. pump-users (251 (180, 340) vs. 322 (263, 406); p = 0.008). Significant univariate correlates of mtDNA-cn: T1D: (positive) HDL-C; (negative) fasting glucose, white cell count (WCC), sVCAM-1, sICAM-1; CON: (negative) WHR (waist-hip-ratio). Detectable C-peptide in T1D increased with lowest-highest mtDNA-cn tertiles (54%, 69%, 79%, p = 0.02). Independent determinants of mtDNA-cn: T1D: (positive) HDL-C; (negative) age, sICAM-1; AUROC 0.71; CON: WCC (negative), never smoking, (positive) female, pulse pressure; AUROC 0.74.
mtDNA-cn is lower in T1D vs. CON, and in T1D kidney disease. In T1D, mtDNA-cn correlates inversely with age and inflammation, and positively with HDL-C, detectable C-peptide and pump use. Further clinical and basic science studies are merited.
Cross-links formed within and between proteins are a major cause of protein dysfunction, and are postulated to drive the accumulation of protein aggregates in some human pathologies. Cross-links can ...be formed from multiple residues and can be reversible (usually sulfur-sulfur bonds) or irreversible (typically carbon-carbon or carbon-heteroatom bonds). Disulfides formed from oxidation of two Cys residues are widespread, with these formed both deliberately, via enzymatic reactions, or as a result of unintended oxidation reactions. We have recently demonstrated that new protein-glutathione mixed disulfides can be formed through oxidation of a protein disulfide to a thiosulfinate, and subsequent reaction of this species with glutathione. Here we investigate whether similar reactions occur between an oxidized protein disulfide, and a Cys residues on a second protein, to give novel protein cross-links. Singlet oxygen (1O2)-mediated oxidation of multiple proteins (α-lactalbumin, lysozyme, beta-2-microglobulin, C-reactive protein), and subsequent incubation with the Cys-containing protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH), generates inter-protein cross-links as detected by SDS-PAGE, immunoblotting and mass spectrometry (MS). The cross-link yield is dependent on the 1O2 concentration, the presence of the original protein disulfide bond, and the free Cys on GAPDH. MS with 18O-labeling has allowed identification of the residues involved in some cases (e.g. Cys25 from the Cys25-Cys80 disulfide in beta-2-microglobulin, with Cys149 or Cys244 of GAPDH). The formation of these cross-links results in a loss of GAPDH enzymatic activity. These data provide ‘proof-of-concept’ for a novel mechanism of protein cross-link formation which may help rationalize the accumulation of cross-linked proteins in multiple human pathologies.
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•Disulfide bonds (DSBs) are critical to protein structure and function.•DSBs are rapidly oxidized by singlet oxygen and other oxidants to reactive intermediates.•These intermediates react with Cys-containing proteins to give new protein-protein cross-links.•This novel disulfide cross-linking pathway affects the functional activity of the proteins.•These cross-links can be diminished by reductants, but this does not repair the DSB damage.
Abstract
To determine whether continuous subcutaneous insulin infusion (CSII) vs. multiple daily injections (MDI) therapy from near-diagnosis of type 1 diabetes is associated with reduced glycaemic ...variability (GV) and altered microRNA (miRNAs) expression. Adolescents (74% male) within 3-months of diabetes diagnosis (n = 27) were randomized to CSII (n = 12) or MDI. HbA1c, 1-5-Anhydroglucitol (1,5-AG), high sensitivity C-peptide and a custom TaqMan qPCR panel of 52 miRNAs were measured at baseline and follow-up (median (LQ-UQ); 535 (519–563) days). There were no significant differences between groups in baseline or follow-up HbA1c or C-peptide, nor baseline miRNAs. Mean ± SD 1,5-AG improved with CSII vs. MDI (3.1 ± 4.1 vs. − 2.2 ± − 7.0 mg/ml respectively,
P
= 0.029). On follow-up 11 miRNAs associated with diabetes vascular complications had altered expression in CSII-users. Early CSII vs. MDI use is associated with lower GV and less adverse vascular-related miRNAs. Relationships with future complications are of interest.
Abstract
The aim of this cross-sectional study was to compare plasma C-peptide presence and levels in people without diabetes (CON) and with Type 1 diabetes and relate C-peptide status to clinical ...factors. In a subset we evaluated 50 microRNAs (miRs) previously implicated in beta-cell death and associations with clinical status and C-peptide levels. Diabetes age of onset was stratified as adult (≥ 18 y.o) or childhood (< 18 y.o.), and diabetes duration was stratified as ≤ 10 years, 10–20 years and > 20 years. Plasma C-peptide was measured by ultrasensitive ELISA. Plasma miRs were quantified using TaqMan probe-primer mix on an OpenArray platform. C-peptide was detectable in 55.3% of (n = 349) people with diabetes, including 64.1% of adults and 34.0% of youth with diabetes,
p
< 0.0001 and in all (n = 253) participants without diabetes (CON). C-peptide levels, when detectable, were lower in the individuals with diabetes than in the CON group median lower quartile (LQ)–upper quartile (UQ) 5.0 (2.6–28.7) versus 650.9 (401.2–732.4) pmol/L respectively,
p
< 0.0001 and lower in childhood versus adult-onset diabetes median (LQ–UQ) 4.2 (2.6–12.2) pmol/L vs. 8.0 (2.3–80.5) pmol/L,
p
= 0.02, respectively. In the childhood-onset group more people with longer diabetes duration (> 20 years) had detectable C-peptide (60%) than in those with shorter diabetes duration (39%,
p
for trend < 0.05).
Nine miRs significantly correlated with detectable C-peptide levels in people with diabetes and 16 miRs correlated with C-peptide levels in CON. Our cross-sectional study results are supportive of (a) greater beta-cell function loss in younger onset Type 1 diabetes; (b) persistent insulin secretion in adult-onset diabetes and possibly regenerative secretion in childhood-onset long diabetes duration; and (c) relationships of C-peptide levels with circulating miRs. Confirmatory clinical studies and related basic science studies are merited.
•Telomere length increased over time in 40% of people with type 1 diabetes.•Baseline telomere length correlated inversely with telomere length change over time.•Antihypertensive and lipid-lowering ...drugs are associated with telomere length change.•Obesity and kidney function correlate with telomere length change.
We aimed to determine how white blood cell (WBC) telomeres and telomere length change over time are associated with health status in type 1 diabetes.
Relative telomere length (rTL) was measured in WBC DNA from two time-points (median 6.8 years apart) in 618 individuals from the Finnish Diabetic Nephropathy Study by quantitative PCR, with interassay CV ≤ 4%.
Baseline rTL correlated inversely with age and was shorter in men. Individuals in the shortest vs. longest rTL tertile had adverse cardiometabolic profiles, worse renal function, and were prescribed more antihypertensive and lipid-lowering drugs. While overall rTL tended to decrease during the median 6.8-years of follow-up, telomeres shortened in 55.3% of subjects, lengthened in 40.0%, and did not change in 4.7%. Baseline rTL correlated inversely with rTL change. Telomere lengthening was associated with higher HDL-Cholesterol (HDL-C), HDL-C/ApoA1, and with antihypertensive drug and (inversely) with lipid-lowering drug commencement during follow-up. Correlates of rTL percentage change per-annum (adjusted model) were baseline BMI, eGFR, previous retinal laser treatment, HDL-C, and HDL-C/ApoA1.
Telomere length measurements may facilitate the treatment and monitoring of the health status of individuals with type 1 diabetes.
Inter- and intra-molecular crosslinks can generate protein dysfunction, and are associated with protein aggregate accumulation in aged and diseased tissues. Crosslinks formed between multiple amino ...acid side chains can be reversible or irreversible. Disulfides formed either enzymatically, or as a result of oxidant-mediated reactions, are a major class of reversible crosslinks. Whilst these are commonly generated via oxidation of Cys thiol groups, they are also formed by ‘oxidant-mediated thiol-disulfide reactions’ via initial disulfide oxidation to a thiosulfinate or zwitterionic peroxide, and subsequent reaction with another thiol including those on other proteins. This generates new intermolecular protein-protein crosslinks. Here we demonstrate that photooxidation, or reaction with the biological oxidants HOCl and ONOOH, of the single disulfide present in the major human plasma inflammatory protein, C-reactive protein (CRP) can give rise to reversible disulfide bond formation with human serum albumin (HSA). This occurs in an oxidant dose-, or illumination-time-, dependent manner. These CRP-HSA crosslinks are formed both in isolated protein systems, and in fresh human plasma samples containing high, but not low, levels of CRP. The inter-protein crosslinks which involve Cys36 of CRP and Cys34 of HSA, have been detected by both immunoblotting and mass spectrometry (MS). The yield of protein-protein crosslinks depends on the nature and extent of oxidant exposure, and can be reversed by dithiothreitol and tris(2-carboxyethyl)phosphine hydrochloride. These data indicate that oxidation of disulfide bonds in proteins can be a source of novel inter-protein crosslinks, which may help rationalize the accumulation of crosslinked proteins in aged and diseased tissues.
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•C-reactive protein (CRP) is a major acute phase inflammatory protein in human plasma.•Oxidation of the single Cys36-Cys97 disulfide in CRP generates reactive intermediates.•The oxidized disulfide reacts with Cys34 of human serum albumin to forms a new crosslink.•The inter-protein CRP-HSA crosslink has been characterized by immunoblotting and LS-MS/MS.•This novel crosslink may be a long-lived plasma marker of inflammation-induced damage.
The phosphoinositide 3-kinase (PI3K)-Akt network is tightly controlled by feedback mechanisms that regulate signal flow and ensure signal fidelity. A rapid overshoot in insulin-stimulated recruitment ...of Akt to the plasma membrane has previously been reported, which is indicative of negative feedback operating on acute timescales. Here, we show that Akt itself engages this negative feedback by phosphorylating insulin receptor substrate (IRS) 1 and 2 on a number of residues. Phosphorylation results in the depletion of plasma membrane-localised IRS1/2, reducing the pool available for interaction with the insulin receptor. Together these events limit plasma membrane-associated PI3K and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) synthesis. We identified two Akt-dependent phosphorylation sites in IRS2 at S306 (S303 in mouse) and S577 (S573 in mouse) that are key drivers of this negative feedback. These findings establish a novel mechanism by which the kinase Akt acutely controls PIP3 abundance, through post-translational modification of the IRS scaffold.
Systems genetics has begun to tackle the complexity of insulin resistance by capitalising on computational advances to study high-diversity populations. 'Diversity Outbred in Australia (DOz)' is a ...population of genetically unique mice with profound metabolic heterogeneity. We leveraged this variance to explore skeletal muscle's contribution to whole-body insulin action through metabolic phenotyping and skeletal muscle proteomics of 215 DOz mice. Linear modelling identified 553 proteins that associated with whole-body insulin sensitivity (Matsuda Index) including regulators of endocytosis and muscle proteostasis. To enrich for causality, we refined this network by focusing on negatively associated, genetically regulated proteins, resulting in a 76-protein fingerprint of insulin resistance. We sought to perturb this network and restore insulin action with small molecules by integrating the Broad Institute Connectivity Map platform and in vitro assays of insulin action using the Prestwick chemical library. These complementary approaches identified the antibiotic thiostrepton as an insulin resistance reversal agent. Subsequent validation in ex vivo insulin-resistant mouse muscle and palmitate-induced insulin-resistant myotubes demonstrated potent insulin action restoration, potentially via upregulation of glycolysis. This work demonstrates the value of a drug-centric framework to validate systems-level analysis by identifying potential therapeutics for insulin resistance.
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